Human Malignant Mesothelioma Cell Growth: Activation of Janus Kinase 2 and Signal Transducer and Activator of Transcription La for Inhibition by Interferon-Y!

Home , FER (gene), Janus kinase 2

(CANCER RESEARCH 58. 840-847. February 15. 1998] Human Malignant Mesothelioma Cell Growth: Activation of Janus Kinase 2 and Signal Transducer and Activator of Transcription la for Inhibition by Interferon-y!

Annie Buard, Claire Vivo, Isabelle Monnet, Christian Boutin, Yannick Pilatte, and Marie-Claude Jaurand2

Institut National de la SantÃ©et de la Recherche MÃ©dicaleU139, FacultÃ©de MÃ©decine{A. B., C. V., M-C. J., Y. P.], Ser\'ice de Pneumologie, Centre Hospitalier Intercommunal de CrÃ©teil11.M./. V4010 CrÃ©leilCedex, and HÃ´pital de la Conception, Marseille 1C. B.Â¡.France

ABSTRACT methods are currently being developed to improve the effects of cytokines in tumors treatment. Gene therapy using vectors that encode Intrapleural injections of recombinant human IFN-y have shown some for cytokines genes has drawn particular interest (12). However, as it efficacy in reducing tumor growth in early stages of diffuse malignant was recently emphasized (13), it is very important to document the mesothelioma i DMM i. Here, we have addressed the potential therapeutic effect of IFN-y in DMM by investigating the activation of the JAK/STAT mechanisms of action of these therapeutic agents at the cellular level signaling pathway in seven human mesothelioma cell lines (HMCLs) that to understand the outcomes in clinical studies. were differentially responsive to the antiproliferative activity of IFN-y. Although modulation of the immune response likely accounts for We showed that janus kinase 2 (JAK2) and signal transducer and activa part of the antitumoral effects of IFN-y in DMM (4, 14), we have tor of transcription 1 (STATI) were phosphorylated on Ivrosine residues demonstrated previously that IFN-y elicited a direct antiproliferative within 15 min in all the HMCLs in which IFN-y (500 units/ml) inhibited effect in vitro in 50% of well-characterized HMCLs isolated from proliferation. In addition, STATI binding activity to the gamma-activated DMM patients (15). Further examination of the mechanisms by which sites DNA sequence was detected within 15 min in electrophoretic mobil IFN-y exerts its antitumoral action in HMCLs indicated that the ity-shift assay analysis, and IFN regulatory factor-1 RNA expression was observed within 6 h in the more responsive cells (72.7-95.2% inhibition of differential growth-suppressive effect of IFN-y in HMCLs was inde DNA synthesis after 72 h of treatment). Conversely, in several HMCLs, pendent of the activation of nitric oxide synthase and indoleamine absent or limited growth suppressive effect (less than 22% inhibition of 2,3-dioxygenase (14-16). Because the IFN-yR had been found to be DNA synthesis) was associated with alterations in expression or activation expressed in all these cell lines at both the RNA and protein levels (14, of JAK2 or STATI or, downstream, with low induction of IFN regulatory 16), here, we examined the capacity of IFN-y to activate its signaling factor-1 RNA expression and/or STATI protein expression following pathway and whether molecular alterations were present downstream IFN-y treatment. These data suggest that at least part of the IFN-y effect of the receptor in unresponsive cell lines. on proliferation of HMCLs is mediated directly through activation of the Elements of the signal transduction pathway that are specific to JAK/STAT1 signaling pathway, and it could account for the antitumoral IFN-y have been identified previously using mutagenized cells (17- activity reported in DMM patients treated with IFN-y. 19). Briefly, binding of IFN-y to its heterodimeric receptor induces oligomerization of the receptor subunits and causes phosphorylation INTRODUCTION of the IFN-yR a chain by two associated tyrosine kinases, JAK1 and DMM' is a severe primary tumor of the pleura that is frequently JAK2. The activity of JAK1 and JAK2 results in recruitment and phosphorylation of the latent cytoplasmic transcription factor p91/ associated with asbestos exposure, and it has poor prognosis (1, 2). STAT la, which is then dimerized and translocated to the nucleus, Current therapies, including surgery, chemotherapy, and radiotherapy, where it binds to GAS consensus sequences that present in the have been unsuccessful in improving patient survival. Nevertheless, promoter of IFN-y-activated genes. immunotherapy has emerged as an alternative therapy since inocula Biological models containing mutations in the IFN-y-signaling tion of recombinant human IFN-y in the pleural cavity of mesotheli pathway have brought into light the roles of JAK l, JAK2, and STATI oma patients was shown to be effective in reducing progression of proteins in IFN-y-mediated effects, such as immune and antiviral early stages of malignant mesothelioma (3, 4). Clinical trials with other cytokines, such as IFN-a2a and 1L-2, were also reported to responses (7, 8). For example, STATI-deficient mice exhibited a lack of IFN-y-induced responses, such as gene activation, induction of result in partial responses (5, 6). In addition to immunomodulatory activity, IFN-y is thought to act directly on cell growth, as demon immunomodulatory molecules, and antimicrobial and antiviral activ strated by the antiproliferative effect that was observed in various cell ities (20, 21). Similarly, mutant cells defective in either JAK1 or types, including epithelial and hematopoietic cells (7-11). These JAK2 were found to be unresponsive to IFN-y (18, 22). More re cently, expression of a kinase-negative mutant of JAK2 resulted in a results suggest that cytokine therapy might prove successful in some particularly drug-resistant diseases, such as DMM. New therapeutic blockade of IFN-y-inducible gene expression (23). Less is known regarding genes involved in IFN-y-mediated regulation of cell

Received 10/9/97; accepted 12/11/97. growth. Nonetheless, recent data have supported a direct link between The costs of publication of this article were defrayed in part by the payment of page activation of the IFN-y-signaling pathway and control of proliferation charges. This article must therefore be hereby marked advertisement in accordance with through activation of STATI (24). The mutagenized human cell line 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Institut National de la SantÃ©etde la Recherche MÃ©dicale U3A, which does not express STATI, is not growth inhibited by funds, by a Legs Poix subvention, and by the Association pour la Recherche sur le Cancer IFN-y, but growth response was restored by transfection of STATI (to A. B.). 2 To whom requests for reprints should be addressed, at Institut National de la SantÃ© (24). However, no such alterations have been found naturally so far in el de la Recherche MÃ©dicaleU139. FacultÃ©deMÃ©decine.8. rue du GÃ©nÃ©ralSarrau,94010 human cells isolated from tumors. CrÃ©teilCedex. France. Phone: 33-1-49-81-36-66; Fax: 33-1-49-81-35-33; E-mail: No prior data have been previously reported with regard to the jaurand Ã•?1im3.inserm.fr. 1The abbreviations used are: DMM. diffuse malignant mesothelioma: IL. interleukin; mechanisms of IFN-y-mediated growth inhibition in HMCLs. Our in HMCL. human mcsothelia cell line; IFN-yR. IFN--y receptor; IRF. IFN regulatory factor; vitro data indicated that several cell lines were resistant or poorly JAK. janus kinase: STAT, signal transducer and activator of transcription; GAS. gamma- activated sites; ECL. enhanced chemiluminescence; EMSA. electrophoretic mobility-shift responsive to the cytostatic effect of IFN-y (15, 16). Therefore, we assay; TCA. trichloroacetic acid. investigated whether JAK2 and STATI were activated in response to 840

Table 1 Patient and cell line data (Calbiochem, France Biochem, Meudon, France). Blots were developed by ECL (Amersham, Les Ulis, France). Membranes were reblotted with anti- linesCytological STATl, anti-JAK2, or anti-IFN-yR, to confirm that equal amounts of immu- Case of staging"IV noprecipitates were loaded in all lanes. no.1 exposureYes type*E specimen'PI type*E Scanning densitometry was performed using ImageAssistant (Omnipage Professional, Version 5.0, Caere Inc., MÃ¼nchen,Germany), and quantifications 2 MF 65 IV Unknown E Pe COS E 3 65 Irv,iUaHaTumorsAsbestosYes E Pe FER E were done with Scan Analysis (Version 2.0, Biosoft, Cambridge, United 4 M 65 Yes EdEEOrigin PI HIB M Kingdom). 5 FMMAge(yr)7974 Unknown Pe HUO E EMSA. Cells were incubated with or without 500 units/ml IFN-y for 15 6 71 Yes PI MORC E min at 37Â°C.After washes with ice-cold PBS, cells were scraped in 1 ml of 7SexM 65PalienisMesotheliomaPossibleHistolÃ³gica! PIGelCodeCORORAV11 E " See Ref. 4 for patient staging information. PBS. Whole cell extracts were prepared by resuspending the pelleted cells in a small volume of lysis buffer [10 mM HEPES (pH 7.9), 0.1% Nonidet P-40, E, epithelial; M. mixed. ' PI, pleural biopsy; Pe. pleural effusion. 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol. 140 mM NaCl, 5 /ig/ml leupeptin, d â€”.not available. 5 /ig/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Concentration of NaCl in the lysates was brought to 400 mM by addition of 5 mM NaCl. After l h of incubation on ice, the mixture was IFN-7 treatment in a series of HMCLs and whether activation of the centrifuged at 18,000 X g for 15 min at 4Â°C.Supernatants were collected and kept at -80Â°C until use. The oligonucleotide probe (5'-CTAGATTTCCCA- signaling pathway correlated with differential sensitivity in response GAAAAGGAACATGATGAAT-3') for the GAS present in the promoter to IFN-y-antiproliferative effect. We show that expression and/or of the high-affinity Fey receptor gene was a generous gift from Dr. van activation of these major components of the transduction pathway Weyenbergh (Institut National de la SantÃ©etde la Recherche MÃ©dicaleU365, were altered in HMCLs that were not growth inhibited by IFN-7. Our Institut Curie, Paris, France). The fragment was end-labeled with [a-12P]dCTP findings are significant in the context of the developing clinical use of using the Ready-To-Go DNA-labeling kit (Pharmacia Biotech) and purified on IFN-y as immunotherapy for human malignant mesothelioma and G50 Sephadex columns. Protein extracts (10-15 /xg) were incubated for 30 might be relevant to other human pathologies. min on ice with the radiolabeled probe (60,000 cpm) in a final volume of 16 p.\ of 1x binding buffer [5x: 50 mM HEPES (pH 7.9), 25 mM Tris-Cl, 2.5 mM MATERIALS AND METHODS DTT, 1 mM EDTA, 50% glycerol, and 500 Â¿ig/mlBSA], containing 1 >Â¿gof sonicated salmon sperm DNA. Competition assays were performed by adding Materials. Purified recombinant human IFN-y (specific activity, 3 X IO7 a 100-fold molar excess of the unlabeled oligonucleotide. Supershift experi units/mg) was a generous gift from Boehringer Ingelheim (Reims. France). ments were performed by incubating protein extracts with 1 /xg of anti-STATl Anti-STATl and anti-JAK2 antibodies were purchased from Santa Cruz Bio antibody for I h at 4Â°C,prior to the addition of the radiolabeled oligonucleo technology (TEBU s.a., Paris, France). Antiphosphotyrosine antibody (4G10) tide. Additional control consisted in preincubating the reaction with 1 /xg of antibody unrelated to STATI (anti-p21WAF ' antibody was used; Transduction was provided by Upstate Biotechnology, Inc. (Euromedex, Souffelweyer- sheim, France). Anti-IFN-yR antibody was obtained from Genzyme (Paris, Laboratory, Medgene Science, Pantin, France). The DNA-prolein complexes France). were separated from the free probe on 5% nondenaturing polyacrylamide gels Human Mesothelioma Cell Lines. Specimens were obtained from seven in 1x TGE buffer (50 mM Tris-Cl, 0.2 Mglycine, and 2 mM EDTA). The dried gels were exposed at -80Â°C. patients with confirmed malignant mesothelioma of the pleura and were provided by the Departments of Pulmonary Medicine of HÃ´pital de la Con Northern Blot Analysis. Total RNA was prepared using the standard ception (Marseille, France) and of Centre Hospitalier Intercommunal (CrÃ©teil, guanidium-isothiocyanate method. Total RNA extracts prepared from HeLa France). Mesothelioma cell lines were then established from pleural effusions cells, treated with or without IFN-y, were kindly provided by Dr. Alcaide or tumors, as described previously (15). The clinical data and cell line char (Institut National de la SantÃ©et de la Recherche MÃ©dicaleU276, Institut acteristics are presented in Table 1. Cell lines were routinely maintained in Pasteur, Paris, France) and were used as positive controls. Twenty or 10 fig of RPMI 1640 (Life Technologies, Inc., Cergy-Pontoise, France), supplemented HeLa cells extracts were resolved on a denaturing 1.2% agarose gel containing with 10% fetal bovine serum (Life Technologies, Inc.), 5 mM glutamine, 50 0.4 M formaldehyde, transferred to a nylon membrane, and hybridized over night at 65Â°Cto [a-'2P]dCTP-labeled probe (2 x 10fi cpm/ml). cDNA probe /xg/ml streptomycin, and 50 units/ml penicillin (ATGC Biotechnologie, Noisy- Le-Grand, France) and buffered with 10 mM HEPES (ATGC). The HMCLs was a 1-kb fragment of the IRF-l gene (provided by Dr. H. Uedo, Hyogo, used here have been studied previously with regard to their immunocytochem- Japan). Probe was labeled by random priming using the Ready-To-Go labeling ical characteristics (14, 15, 25). Here, HMCLs (passages 6-20) were treated kit (Pharmacia Biotech). Following hybridization, filters were washed twice in with 500 units/ml for 15 min, or as indicated. ix SSC-0.1% SDS for 10 min at room temperature and once in O.lx Immunoprecipitations and Western Blots. Cells were plated at 2 X Id' SSC-0.1% SDS for 10 min at 65Â°C. cells/cm2 in 75-cm2 flasks, and medium was changed the following day. Two Thymidine Incorporation Assay. The antiproliferative effect of IFN-y days latter (50% confluency), cells were either left untreated or treated with was assessed on cell lines cultured on 12-well plates. For this assay, cells were 500 units/ml IFN-y for 15 min or as indicated at 37Â°C.Cultures were washed plated at 2 X IO4 cells/cm2 and grown for 24 h in complete medium. Cells twice with ice-cold PBS and scraped in extraction buffer [50 mM Tris-HCl (pH were either treated with 500 units/ml IFN-y or left untreated. After 72 h of treatment, cells were pulsed with ['HJthymidine (5 fxCi/ml; specific activity, 7.4), 150 mM NaCl, 1% Nonidet P-40, 50 mM sodium fluoride, I mM EDTA, 1 mM sodium orthovanadate, 10 /xg/ml leupeptin, 10 /Â¿g/mlaprotinin, and I 40-60 mCi/ml; ICN Pharmaceuticals, Inc., Orsay, France) for 60 min. Cells were washed three times with Dulbecco's PBS (Seromed, Berlin, Germany) HIM phenylmethylsulfonyl fluoridel. Cells were further lysed by repeated passages through a 21-gauge needle, and lysates were cleared by centrifugaron and incubated with I ml of ice-cold 10% TCA for 10 min. The TCA- at 16,000 X g for 15 min at 4Â°C.The supernatants were precleared with protein precipitable fraction was solubilized in 200 Â¡uof 0.2 M NaOH-1% SDS, and A-Sepharose beads (Pharmacia Biotech, Orsay, France) with rocking for 30 [3H]thymidine incorporation was determined with a LS6000SC scintillation min at 4Â°C.Protein concentrations were determined using the DC protein assay counter (Beckman, Gagny. France). [3H]Thymidine incorporation in triplicates (Bio-Rad, Ivry-sur-Seine, France) and normalized by addition of extraction of IFN-y-treated cultures was expressed as a percentage of incorporation in buffer. Lysates were incubated for 2 h at 4Â°Cwith anti-STATl antibody, untreated control cultures. anti-JAK2, or anti-IFN-yR. Immunocomplexes were collected on protein A-Sepharose and washed four times with extraction buffer. Immunoprecipi- RESULTS tates were analyzed by SDS-PAGE (7.5% gel), followed by transfer onto Immobilon-P membranes (Millipore, Paris, France). Membranes were blocked Activation of JAK2 in Response to IFN-y Treatment in with 5% nonfat dry milk in PBS-0.05% Tween and blotted with antiphospho- HMCLs. We have reported previously that HMCLs were differen tyrosine (4G10), followed by horseradish peroxidase-labeled antimouse IgG tially responsive to IFN-y antiproliferative activity (15). Because the 841

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. IFN-y SIGNALING PATHWAY IN HUMAN MESOTHELJOMA CELL LINES lack of response to IFN-y in HMCLs was not related to the absence of receptor (14, 16), we investigated whether differential sensitivity to IP: the antiproliferative activity of IFN-y was related to molecular dif Blot: ferences in the capacity to activate the major downstream components of the IFN-y signaling pathway. The central role played by JAK2 in Cell Lines: the IFN-y-signaling pathway led us to first analyze its activation in Time on IFN-y:P-IFNyR 2' 10' 15'FER- 2' 5' 15'MW various HMCLs treated with IFN-y. â€” 116 With the exception of one cell line, FER, phosphorylation of JAK2 -*-anti-IFNyRanti-P-TyrMORC- on tyrosine residues was detected in all of the HMCLs tested follow â€” 84 ing a 15-min treatment with IFN-y (Fig. 1 top, and data not shown). In the cell line FER, no phosphorylation was observed in response to anti-IFNyR IFN-y treatment (Fig. 1, left), and reblotting of the same membrane Blot: with an anti-JAK2 antibody indicated that the protein could not be detected in these cells (Fig. 1, bottom). The presence of a defect in the IFNyR -*- IFN-y signaling pathway in the cell line FER was confirmed by examining phosphorylation of IFN-yR in response to IFN-y treatment Fig. 2. Tyrosine phosphorylation of the IFN-yR by IFN--y in HMCLs. Cells were either (Fig. 2). Lysates from the cell lines MORC and FER, treated with or left untreated ( - ) or treated with 500 units/ml IFN-y for the indicated times at 37Â°C.Total without 500 units/ml IFN-y, were subjected to immunoprecipitation cell lysates were precleared. and protein was immunoprecipitated with 3 /Ag of antibody with an anti-IFN-yR antibody, followed by immunoblotting with an to the IFN-yR, as described in "Materials and Methods." Immunoprecipitates were analyzed by Western blotting first with an antiphosphotyrosine antibody (4G10; top) and antiphosphotyrosine (Fig. 2, top). Tyrosine phosphorylation of the then with an antibody to IFN-yR (bottom). Blots were revealed with ECL. Arrows, receptor was detected within 2 min in the cell line MORC, with a peak phosphorylated IFN-yR (top) and the IFN-yR protein that was immunoprecipitated at 5 min (data not shown), and it was still detected after 15 min. In (bottom). contrast, no modifications were observed in the cell line FER treated with IFN-y. Reblotting of membranes indicated that similar amounts of the protein were immunoprecipitated in each lane (Fig. 2, bottom). Therefore, we next analyzed phosphorylation of STATI and IFN-y- Absence of phosphorylation of the IFN-yR in the cell line FER was induced STATl-DNA binding activity to assess whether STATI was consistent with the lack of phosphorylation of JAK2 in these cells functional in HMCLs. Cellular extracts were subjected to immuno because a defect in the expression and/or functioning of JAK2 would precipitation with an anti-STATl antibody, followed by SDS-PAGE prevent activation of JAK l, thereby impairing receptor phosphoryla and blotting with antiphosphotyrosine. STATI was activated by ty tion (23). Thus, in all but one HMCLs tested, IFN-y treatment resulted rosine phosphorylation within 15 min following addition of IFN-y in in tyrosine phosphorylation of JAK2, irrespective of their response to most of the cell lines, i.e., HIB, HUO, RAV, COS, and MORC (Fig. IFN-y growth-suppressive effect. 3 top). In contrast, tyrosine phosphorylation of STATI was very low Activation of STATI in IFN-y-treated HMCLs. The importance in CORO and absent in FER (Fig. 3). Blots were reprobed with of STATI activity in the IFN-y biological response has been sup anti-STATl antibody to ascertain that comparable amounts of STATI ported by numerous in vivo and in vitro experiments (20, 21, 24, 26). were immunoprecipitated from treated and untreated cultures (Fig. 3, bottom). To determine whether the phosphorylated STATI was functionally active, we examined the capacity of STATI to bind to the GAS IP anti-JAK2 anti-JAK2 consensus sequence. Detection of STATI-DNA-binding activity in EMS A analysis after 15 min of IFN-y treatment confirmed that Blot anti-P-Tyr anti-P-Tyr STATI was activated in the cell lines MORC (Fig. 4A, Lanes 1-7), COS (Fig. 4B, Lanes 1-7). HIB (Fig. 4C, Lanes 1-4), HUO (Fig. 4C, Cell Lines FER MORC HIB COS Lanes 5-8), and RAV (Fig. 4C. Lanes 9-15). Although this was not IFN-y readily detectable, some of the STATI protein was able to bind to the GAS DNA sequence in the cell line CORO treated with IFN-y (Fig. 4B, Lane 9). In contrast, STATl-DNA binding activity was absent in P-JAK2 -Â»- FER (Fig. 4A, Lanes 8-13). Excess unlabeled GAS oligonucleotide competed with formation of labeled GAS-STATI complexes, as as sessed by reduced intensity of the band detected in MORC (Fig. 4A, compare Lanes 2 and 4), HIB (Fig. 4C, compare Lanes 2 and 3), HUO Blot anti-JAK2 anti-JAK2 (Fig. 4C, compare Lanes 6 and 7), and RAV (Fig. 4C, compare Lanes 10 and 12). Specificity of the GAS DNA binding was verified by preincubating lysates with an anti-STATl antibody, which altered JAK2 â€” migration of GAS-protein complexes (supershift) in MORC (Fig. 4A, compare Lanes 2 and 6) and COS (Fig. 4B, compare Lanes 2 and 6). Although the anti-STATl-induced supershift was not as clearly de Fig. 1. Tyrosine phosphorylation of JAK2 by IFN-y in HMCLs. Cells were either left untreated (-) or treated with 500 U/ml IFN-y ( + ) for 15 min at 37Â°C.Total cell lysates tected in other cell lines, including HIB (Fig. 4C, Lane 4), HUO (Fig. were precleared, and 500-800 fig of protein were immunoprecipitated with 3 Â¿igof 4C, Lane 8), and RAV (Fig. 4C, Lane 14), intensity of the GAS- antibody to JAK2. as described in "Materials and Methods." JAK2 immunoprecipitates STATI complexes was reduced in the presence of the antibody. In were analyzed by Western blotting first with an antiphosphotyrosine antibody (4G10; top) and then with the anti-JAK2 antibody to verify that equal amounts of protein were contrast, addition of an antibody unrelated to STATI had no effect on immunoprecipitated in control and treated cultures (bottom). Blots were revealed with formation and migration of the STATl-DNA complexes, as shown in ECL. Arrow (bottom). JAK2: corresponds lo the band which is phosphorylated in the IFN-y-lrealed samples (lop. P-JAK2). The additional bands detected with the JAK2 Fig. 4A (Lane 7, MORC), Fig. 4B (Lane 7, COS), and Fig. 4C (Lane antiserum in COS and CHAN were nonspecific and were not consistently detected. Â¡5,RAV), indicating that this DNA-binding activity was specific of 842

IP: anti-STAT1aHIB Blot: Cell Lines FER- CORO-+-+-RAV MORC- Fig. 3. Tyrosine phosphorylation of STATI by IFN-y in HMCLs. Cells were incubated in the IFN-Y absence ( - ) or presence ( + ) of 500 units/ml IFN-y +....~+ -+mmanti-P-TyrHUO+anti-STAT1aCOS + - for 15 min at 37Â°C.Total cell lysates were precleared, and 500-800 /ig of protein were immunoprecipitated with 3 ng of antibody to STATI, as described in "Materials and Methods." STATI im- P-STAT1a munoprecipitales were analyzed by Western blot ting, first with an antiphosphotyrosine antibody (4G10; topi and then with the anti-STATl antibody (bottom}. Blots were revealed with ECL. Blot:

STATIa -Â»- ~ ;

STATI in HMCLs extracts. The absent or reduced DNA-binding (Fig. 3) and Western blot analysis of total cell lysates (data not shown) activity that was observed upon IFN-y treatment in the cell lines FER indicated that all of the HMCLs expressed constitutive amounts of (Fig. 4A, Lanes 8-ÃŒ3)and CORO (Fig. 4B, Lanes 8-12), respectively, STATI, despite differences in expression levels. As shown in Fig. 6/4 suggested that STATI was not activated or not fully activated in these for HIB, IFN-y treatment induced accumulation of STATI protein, HMCLs. which was detectable in HMCLs as early as 9 h after addition of the IFN-y-induced IRF-1 Gene Expression Correlates with Anti- cytokine. In responsive HMCLs, STATI protein expression was dra proliferative Effect in HMCLs. Induction of the immediate early matically enhanced after 24 h and remained elevated (6-15-fold gene IRF-1 expression by transcription factor STATI has been related above basal level in untreated cells) at 72 h (Fig. 6B, left, and Table to some of the downstream effects of IFN-y, including inhibition of 2). In contrast, STATI protein levels was unchanged in the resistant proliferation (8, 27-29). Therefore, we further assessed that STATI cell line FER as compared to untreated cells (Fig. 6B, right). Notably, was functional by analyzing HMCLs for IRF-ÃŒgene expression and a moderate increase (less than 2.5-fold) was detected in the poorly cellular proliferation in response to IFN-y treatment (Fig. 5). Effect of responsive HMCLs (see Table 2), which was consistent with the IFN-y on IRF-1 RNA level was examined in HMCLs by Northern limited growth inhibition measured in the corresponding thymidine blot analysis on total RNA isolated after 6 h of treatment (Fig. 5, A assay (see Fig. 5C). and B). In addition, because the activity of IFN-y on cell growth is detectable after a certain delay following addition of IFN-y, we assessed DNA synthesis in cells treated for 72 h (Fig. 5C). Fig. 5C DISCUSSION represents results of [3H]thymidine incorporation in IFN-y-treated Use of IFN-y in clinical trials has led to encouraging results with cultures expressed as a percentage of incorporation in untreated con trol cultures. Basal level of IRF-1 RNA was extremely low (not DMM patients (3, 4, 14). To determine whether IFN-y might operate detected) in untreated cells (Fig. 5A). Following treatment with IFN-y, through a direct mechanism to reduce tumor growth in DMM patients, accumulation of IRF-1 RNA occurred within 6 h in the cell lines HIB, we have investigated the activation of the JAK/STAT signaling path HUO, and MORC (Fig. 5A) and was associated with a marked way following IFN-y treatment of HMCLs that were differentially inhibition of DNA synthesis (Fig. 5Q. [3H]Thymidine incorporation growth inhibited. As summarized in Table 2, we showed that the was only 4.8, 27.3, and 11.7% of control in HIB, HUO, and MORC, IFN-y-signaling pathway was rapidly activated upon treatment in all respectively, after 72 h of treatment with 500 units/ml IFN-y. Con the HMCLs in which IFN-y inhibited cell proliferation, i.e., HIB, versely, in the cell line FER, in which the IFN-y signaling pathway HUO, and MORC. IFN-yR activation occurred within 2 min and was was altered, no induction of IRF-1 RNA was observed after 6 h of followed by phosphorylation of JAK2 and STATI on tyrosine resi treatment, and DNA synthesis was unaffected after 72 h in the dues in most cell lines. Activation of the IFN-y-signaling pathway presence of IFN-y. In other HMCLs, including COS, CORO, and was also demonstrated by the capacity of transcription factor STATI RAV, induction of IRF-1 RNA expression was associated with a to bind to a consensus DNA sequence using EMSAs and by induction limited effect on proliferation (Fig. 5). In these cell lines, [3H]thymi- of the immediate early gene IRF-1 RNA expression using Northern dine incorporation was 77.1, 77.7, and 79.0% of control, respectively, blot analysis. In these cells, DNA synthesis was inhibited by 72.7- after 72 h of treatment. These results suggested that in HMCLs, 95.2%. Conversely, in cells that were responding poorly to growth inefficient growth control might originate from various levels, i.e., inhibition, we observed impaired activation of key components of the either from altered activation of STATI upstream IRF-1 (as in CORO cellular signaling pathway, such as JAK2 and STATI, or of other and FER) or from defects present downstream IRF-1 (as in COS and downstream transcription factors. For example, absence or low level RAV). of JAK2 in one cell line (FER) and inefficient activation of STATI in IFN-r Induces STATI Protein Expression in Growth-inhibited another (CORO) were associated with absent (FER) or limited HMCLs. To further explore the functional differences between these (CORO) ST ATI-DNA binding activity and IRF-1 induction, and HMCLs, we next examined the IFN-y-mediated induction of STATI these alterations were consistent with the lack of growth-inhibitory protein expression as an additional indicator of the activation of one response. In other HMCLs, the moderate growth inhibition was asso of the IFN-y-responsive genes (19, 29). Immunoprecipitation assays ciated with limited induction of STATI protein following IFN-y 843

1 2 3 4 5 6 7 8 9 10 11 12 13 MORC FER IFNy GAS - - + + + + - - anti-STAT1 - - - - + + + + Ab

Supershift â€¢Â»-

GAS *-

NS *~

B 1 234 5 67 89 10 11 12

COS CORO Fig. 4. Analysis of GAS-binding activity of STATI IFNy in EMSA. Whole cell extracts were prepared from HMCI.s that were left untreated (- ) or treated with 500 GAS units/ml IFN-y ( + ) for 15 min. Extracts (10-15 fig) were incubated on ice for 30 min with (or-32P]dCTP anti-STAT1 end-labeled GAS oligonucleotide corresponding to the sequence present in the high affinity Fc-y receptor gene Ab promoter, and GAS complexes were separated on 5% nondcnaturing polyacrylamide gels, as described in "Materials and Methods." GAS, competition with 100- Supershift â€¢*â€¢ fold molar excess of unlabeled oligonucleotide. Where indicated, cell extracts were incubated for l h at 4Â°C with either I /Ag of STATI antiserum (anti-STATlÃ¬or 1 (Xg of antibody unrelated to STATI (Ab), prior to GAS +~ GAS-binding reaction. NS. migration of a nonspecific DNA-prolcin complex. NS +-

1234 5678 9 10 11 12 13 14 15

HIB HUO RAV IFNy

GAS anti-STAT1 Ab

Supershift â€¢Â»-

GAS *~ NS *- H 844

Cell Lines HeLa HIB HUO COS RAV CORO FER MORC IFN-Y - 28S

Fig. 5. IFN--y induced expression of IRF-1 pre IRF-1 ceded inhibition of [3H]thymidine incorporation in - 18S HMCLs. Ten (HeLa cells) or 20 (HMCLs) fig of total RNA were separated on a 1% agarose gel, transferred onto nylon N+ membrane, and hybridized succes sively with IRF-1 (A) and glyceraldehyde-3-phosphate B dehydrogenase (GAPDH: B) probes that were [a-32P]dCTP-labeled, as described in "Materials and Methods." C, cells (20,000/cm2) were treated with 500 GAPDH units/ml IFN-7 fÂ°r72 h. pHJThymidine incorporation was determined on the TCA-precipitable fraction, as described in "Materials and Methods." and is pre sented as a percentage of incorporation in the match ing control cultures. Columns, mean of triplicates wells from at least three independent experiments; bars, SE.

â€¢â€¢â€¢^â„¢Â«i HIB HUO COS FER MORC

treatment, despite normal upstream activation of JAK2 and STATI absence of induction in class II MHC expression (16), as well as in proteins. indoleamine 2,3-dioxygenase (16) and (2', 5')-oligoadenylate synthe- Our results are in agreement with previous studies which have tase activities (data not shown), in addition to the lack of growth- demonstrated the need for functional JAKs and STAT in IFN-y- suppressive effect in response to IFN-y treatment. Altogether, these mediated responses (8, 23). Mutational experiments have indicated results confirmed that the IFN-y-signaling pathway was blocked at a that defects in the signal transduction pathway could result in altered proximal step in this tumor cell line and suggested that this defect was response (17, 20, 22, 24, 26). More specifically, lack of STATI likely to participate in uncontroled growth. expression has been associated with impaired growth-inhibitory re We showed that the sequence of events triggered by IFN-y sponse in mutagenized cell lines (24, 26). We now show that alter treatment was followed by inhibition of growth in responsive ation of expression and/or activation of JAK2 or STATI was associ HMCLs. Conversely, the absence of JAK2 prevented IFN-y-me- ated with altered growth response to IFN-y in tumor cell lines isolated diated growth control in the resistant cell line FER. However, in from human mesothelioma, and these results are of particular interest three of the HMCLs, COS, CORO, and RAV, inhibition of prolif in the context of mesothelioma therapy. eration was moderate (21-23%, as compared to untreated cells) In one cell line, FER, no tyrosine phosphorylation was detected, despite phosphorylation of JAK2 and STATI. Although lower in and immunoblotting of JAK2 immunoprecipitates indicated that this the cell line CORO, the EMSA analysis ascertained that the ca cell line was probably deficient in JAK2. In addition, although the pacity of phosphorylated STATI to recognize the GAS sequence IFN-yR a chain was expressed in FER, it was not phosphorylated was not impaired in these cells (Fig. 4). Similarly, induction of upon IFN-y treatment. Th'5 contrasted with the rapid phosphorylation IRF-1 RNA expression by IFN-y in all three cell lines reflected the of the IFN-yR that occurred in responsive HMCLs. These observa functionality of the signaling pathway, despite differences between tions are consistent with the sequence of events that have been cell lines (Fig. 5). However, it is noteworthy that the limited described previously in various cell types treated with IFN-y (23, 30, IFN-y-induced growth inhibition in the group of poorly responsive 31). That is, following IFN-y binding to its receptor, the presence of cell lines was associated with a lower increase of the STATI a functional JAK2 is necessary for subsequent activation of JAK1- protein levels (2.5-fold or less after 72 h of treatment: see Table 2), mediated phosphorylation of the IFN-yR a chain and recruitment and as compared to responsive HMCLs. It has been suggested that phosphorylation of STATI. For example, JAK1 or JAK2 kinase- STATI autoregulates the STATI gene (29). Thus, the moderate deficient cell lines displayed partial or absent response to IFN-y (23). induction of STATI protein expression could be considered a good Conversely, complementation with JAK2 tyrosine kinase reversed the indication that STATI was not fully transcriptionally active in phenotype of IFN-y-unresponsive mutant cell lines (18). The require response to IFN-y treatment in the three poorly responsive HM ment of JAK2 for phosphorylation of the receptor, as well as of JAK1 CLs. In this regard, there is increasing evidence that the efficacy of and STATI, has also been recently demonstrated for the growth STATI transcriptional activity depends on additional phosphoryl hormone signaling pathway in JAK2~ mutant cells (32). In addition, ation on one or more serine residues (34-36), as well as cooper specific inhibition of JAK2 tyrosine kinase activity by a tyrphostin ative binding of STATI dimers to GAS sites (37). In a recent derivative resulted in inhibition of DNA synthesis in lymphoblastic study, expression of STATI protein bearing a Ser-727-to-Ala-727 leukemia cell lines (33). Therefore, reduced or missing expression of mutation resulted in complete resistance to IFN-y-antiproliferative JAK2 in the tumor cell line FER is likely to contribute to the activity (26). Similarly, it is possible that moderate growth re interruption of IFN-y-signaling pathway, thereby preventing IFN-y- sponse to IFN-y in some HMCLs might be related to altered mediated responses. In FER, defect in JAK2 correlated with the phosphorylation on serine residues on STATI or to endogenous 845

could affect JAK2 because of the possible involvement of this mol ecule in various other signaling pathways. For instance, JAK.2 be Blot anti-STAT1 comes phosphorylated in response to other members of the cytokine Cell Line HIB family, such as IL-6, IL-3, erythropoietin, granulocyte macrophage colony-stimulating factor, growth hormone, and prolactin (19, 32, 38). TimeonlFNY(h) 0 1 3 9 1824 Similarly, expression and activity of STATI and IRF-1 proteins are STATI -*- regulated by various cytokines and growth factors (19, 38), and they are both part of the mechanisms of growth regulation (27-29). These data suggest that HMCLs with missing or inactive signaling compo nents might also exhibit dysregulated growth control by other mole cules. Conversely, it is also possible that the moderate response to IFN-y in some HMCLs might be the result of contradictory influences of growth regulatory molecules. Indeed, it has been previously shown that HMCLs secreted and responded to numerous cytokines and 1 3 91824 growth factors that stimulate proliferation of mesothelioma cells (39, 40). Thus, it will be of interest to study further the interactions B between IFN-y and these other cytokines and growth factors for activation of STATI and IRF-1 in our series of HMCLs. It remains to Blot anti-STAT1HUO24- anti-STAT1FER24- be determined which factor(s) are responsible for the differences in Cell Lines the activation of STATI and IRF-1 and the resulting altered growth Time (h) response in HMCLs. These observations will be of particular interest IFNy +-48- +72-+â€” +48- +72- + with regard to development of new therapeutical strategies for DMM. To our knowledge, this is the first study that examines activation STAT1 -Â»- of JAK2 and STATI in response to IFN-y treatment in human tumor-derived cell lines. In summary, we showed that activation of the IFN-y-signaling pathway was connected to growth inhibition in i1 * mesothelioma tumor cells and, conversely, that the lack of respon siveness to IFN-y-antiproliferative effect was related to alterations 16 li 12 i in the signaling pathway. Furthermore, exploration of this pathway le LO^ tends to suggest that resistance of HMCLs to antiproliferative 0.5 effects of IFN-y is not commonly related to intrinsic molecular 4 pzB Ã•^U SÂ£ O 0 defects of signaling components (with the exception of one cell 24 48 72 24 48 72 line) but rather to modulation of STATI activation by a mechanism Fig. 6. Effect of IFN-7 treatment on STATI protein expression in HMCLs. Cells were that remains to be clarified. Taken together, our data provide incubated in the absence (-) or presence ( + ) of IFN-y for the indicated times at 37Â°C. Total cell lysates (20 Â»igof protein) were analyzed by Western blotting with an anli- further evidence that efficient activation of both JAK2 and STATI STAT1 antibody, as described in "Materials and Methods." Blots were revealed with is required to achieve optimal IFN-y-antiproliferative effect in ECL. Protein levels were compared by scanning densitometry of the films (bottom). A, HMCLs. The consistent IFN-y-mediated modifications we ob time-dependent accumulation of the STATI protein in the responsive cell line HIB. B. served in HMCLs with regard to IRF-1 gene expression, STATI STATI protein expression in the responsive cell line HUO (left) and in the resistant cell line FER (right). The lower level observed in the 24-h-treated sample in FER was due to protein levels, and DNA synthesis suggested that these effects are lower protein loading in this lane, as assessed by staining with Ponceau red prior to directly coupled to the activation of the JAK/STAT pathway in immunoblotting. All other lanes were equally loaded. these cells. Hence, our data also strongly support the idea that IFN-y acts directly on mesothelioma cell growth and, thereby, GAS sites that would not be fully accessible. Thus, one can substantiate the use of IFN-y for mesothelioma therapy. Further imagine that such a defect would prevent optimal transcriptional characterization of both the responsive and unresponsive HMCLs activation of genes specifically involved in growth control. will be of interest to elucidate the mechanisms of action of IFN--y It is noteworthy that alteration of the IFN-y-signaling pathway in DMM, and it is predictable that enhancing IFN-y sensitivity in

Table 2 Summary of IFN-y-mediated activation of the JAK/STAT signaling pathway in HMCLs

phosphorylation"Cell RNA protein STATI activity expression' expression STATIHIBline JAK2 (GASincrease)* binding) (RNA (fold induction)>10>106.7ND' ResponsivelinesPoorly cell + + *4- HUO+MORC + +CORO + +Â± linesUnresponsiveresponsive cell + Â± ++ COS+RAV + 2.51.61.0 +FERIRF-1 + +-STATI cell lineIFN-7-induced " Phosphorylation was detected by Â¡mmunoprecipitation with antibodies to either anti-JAK2 or anti-STATl, followed by Western blot analysis with an antiphosphotyrosine antibody. STATI activity was assessed by EMSA analysis using a [<2P)dCTP-labeled oligonucleotide corresponding to the GAS sequence in the high-affinity Fey receptor gene promoter. ' IRF-1 gene expression was determined by Northern blot analysis in HMCLs treated for 6 h with IFN-y. J Accumulation of STATI protein in HMCLs treated for 72 h with IFN-y was analyzed by Western blotting on whole cell lysates with an antibody to STATI. Results are expressed as fold induction over expression in untreated cells, as determined by densitometry scanning. e ND. not determined.

846

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. IFN-y SIGNALING PATHWAY IN HUMAN MESOTHELIOMA CELL LINES the poorly responsive HMCLs will be beneficial to clinical treat 21. Meraz, M. A., White. J. M., Sheehan, K. C. F., Bach, E. A., Rodig, S. J., Dighe, A. S., ment of DMM. Kaplan, D. H., Riley. J. K., Greenlund, A. C., Campbell, D., Carver-Moore, K., DuBois, R. N., Clark, R., Ague!, M., and Schreiber, R. D. Targeted disruption of the Stall gene in mice reveals unexpected physiologic specificity in the JAK-STAT REFERENCES signaling pathway. Cell, 84: 431-442, 1996. 22. MÃ¼ller,M., Laxton, C., Briscoe, J., Schindler, C.. Improta, T., Darnell, J. E.. Stark, 1. Antman, K. H. Natural history and epidemiology of malignant mesothelioma. Chest, G. R., and Kerr, I. M. Complementation of a mutant cell line: central role of the p91 103 (Suppl.): 373S-376S, 1993. kDa polypeptide of ISGF3 in the interferon-a and -y signal transduction pathways. 2. Boutin, C, Rey, F., Gouverne!, J., Viallat, J. R., Astoul, P., and Ledoray, V. EMBO J., 12: 4221-4228, 1993. Thoracoscopy in pleural malignant mesothelioma: a prospective study of 188 con 23. Briscoe. J.. Rogers. N. C.. Witthuhn, B. A., Watling. D., Harpur, A. G., Wilks. A. F., secutive patients. Cancer (Phila.), 72: 394-404, 1993. Stark, G. R., Ihle, J. N., and Kerr, I. M. Kinase-negative mutants of JAK1 can sustain 3. Boutin, C., Viallat, J. R., Van Zandwijk, N., Douillard, J-Y., Paillard. J-C., Guerin, interferon--y-inducible gene expression but not an antiviral state. EMBO J.. IS: J-C., Mignot, P., Migueres, J., Varlet, F., Jehan, A., Delepoulle, E., and Brandely, M. 799-809, 1996. Activity of intrapleural recombinant -y-interferon in malignant mesothelioma. Cancer 24. Chin, Y. E., Kitagawa, M., Su. W-C. S.. You, Z-H., Iwamoto, Y., and Fu, X-Y. Cell (Phila.), 67: 2033-2037, 1991. growth arrest and induction of cyclin-dependent kinase inhibitor p21WAF"CIPI me 4. Boutin, C., Nussbaum, E., Monnet, I., Bignon, J., Vanderschueren, R., Guerin, J-C., diated by STATI. Science (Washington DC), 272: 719-722, 1996. Menard, O., Mignot, P., Dabouis, G., and Douillard, J-Y. Intrapleural treatment with 25. Zeng, L., Fleury-Feith, J., Monnet, I., Boutin, C., Bignon, J., and Jaurand, M-C. recombinant y-interferon in early stage malignant pleural mesothelioma. Cancer Immunocytochemical characterization of cell lines from human malignant mesothe (Phila.), 74: 2460-2467, 1994. lioma. Hum. Pathol., 25: 227-234. 1994. 5. Christmas, T. I., Manning, L. S., Garlepp, M. J., Musk, A. W., and Robinson, B. W. S. 26. Bromberg. J. F.. Horvath, C. M.. Wen, Z., Schreiber, R. D., and Darnell, J. E. Effect of interferon-a2a on malignant mesothelioma. J. Interferon Res., 13: 9-12, Transcriptionally active Stall is required for the antiproliferative effects of both 1993. interferon a and interferon y. Proc. Nati. Acad. Sci. USA. 93: 7673-7678. 19%. 6. Astoul, P., Viallat, J. R., Laurent, J. C., Brandely, M., and Boutin, C. Intrapleural 27. Harada, H., Kitagawa, M.. Tanaka. N., Yamamoto. H., Harada, K., Ishihara, M., and recombinant IL-2 in passive immunotherapy for malignant pleural effusion. Chest, Taniguchi, T. Anti-oncogenic and oncogenic potential of interferon regulatory fac- 103: 209-213, 1993. tors-1 and -2. Science (Washington DC), 259: 971-974, 1993. 7. Farrar, M. A., and Schreiber, R. D. The molecular cell biology of interferon-y and its 28. Kirchhoff. S.. Schaper, F., and Hauser, H. Interferon regulatory factor 1 (IRF-1) receptor. Annu. Rev. Immunol.. //: 571-611, 1993. 8. Dilliâ„¢. A. Interferon-y: biology and role in pathogenesis. Adv. Immunol., 62: mediates cell growth inhibition by transactivation of downstream target genes. Nu 61-130, 1996. cleic Acids Res., 21: 2881-2889. 1993. 9. Kimchi, A. Cytokine triggered molecular pathways that control cell cycle arrest. J. 29. Pine, R., Canova, A., and Schindler, C. Tyrosine phosphorylated p91 binds to a single Cell. Biochem., JO: 1-9, 1992. element in the ISGF2/IRF-1 promoter to mediate induction by IFNa and IFNy, and is likely to autoregulate the p91 gene. EMBO J., 13: 158-167, 1994. 10. Saunders, N. A., Smith, R. J.. and Jetten, A. M. Differential responsiveness of human bronchial epithelial cells, lung carcinoma cells, and bronchial fibroblasts to interfer- 30. Bach, E. A.. Tanner, J. W.. Marsters, S.. Ashkenazi. A.. Aguel, M.. Shaw, A. S., and on-7 in vitro. Am. J. Respir. Cell Mol. Biol., //: 147-152, 1994. Schreiber, R. D. Ligand-induced assembly and activation of the y interferon receptor 11. Harvat, B. L., and Jetten, A. M. y-Interferon induces an irreversible growth arrest in in intact cells. Mol. Cell. Biol., 16: 3214-3221, 1996. mid-G | in mammary epithelial cells which correlates with a block in hyperphospho- 31. Kotenko, S. V., Izotova, L. S., Pollack, B. P., Mariano, T. M.. Donnelly. R. J., rylation of retinoblastoma. Cell Growth Differ., 7: 289-300, 1996. Muthukumaran, G., Cook, J. R.. Garotta, G.. Silvennoinen. O., Ihle. J. N., and Pestka, 12. Marcel, T., and Grausz. J. D. The TMC worldwide gene therapy enrollment repon S. Interaction between the components of the interferon y receptor complex. J. Biol. (June 1996). Hum. Gene Ther., 7: 2025-2046, 1996. Chem., 270: 20915-20921. 1995. 13. Ross, G., Erickson, R., Knorr, D., Motulsky, A. G.. Parkman, R., Samulski, J., Straus, 32. Han, Y., Leaman, D. W., Watling, D., Rogers, N. C., Groner, B., Kerr, I. M., Wood, S. E., and Smith. B. R. Gene therapy in the United States: a five-year status report. W. I., and Stark. G. R. Participation of JAK and STAT proteins in growth hormone- Hum. Gene Ther., 7: 1781-1790, 1996. induced signaling. J. Biol. Chem., 277: 5947-5952, 1996. 14. Monti, G., Jaurand, M-C., Monnet, I., ChrÃ©tien,P., Saint-Etienne, L., Zeng, L., 33. Meydan, N.. Grunberger, T., Dadi, H.. Shahar. M.. Arpaia, E.. Lapidot, Z., Leeder. Portier, A.. Devillier, P., Galanaud, P.. Bignon, J., and Emilie, E. Intrapleural J. S., Freedman, M., Cohen, A., Gazit, A., Levitzki, A., and Roifman, C. M. Inhibition production of Â¡nterleukin6 during mesothelioma, and its modulation by -y-interferon of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature (Lond.), 379: 645-648, treatment. Cancer Res., 54: 4419-4423, 1994. 1996. 15. Zeng, L., Buard, A., Monnet, I., Boutin, C., Fleury, J., Saint-Etienne, L., Brochard, P., 34. Eilers, A., Georgellis, D.. Klose, B., Schindler. C.. Ziemiecki. A.. Harpur. A. G., Bignon, J., and Jaurand, M-C. In vitro effects of recombinant human Â¡nterferony on Wilks. A. F.. and Decker, T. Differentiation-regulated serine phosphorylation of human mesothelial cell lines. Int. J. Cancer, 55: 515-520, 1993. STATI promotes GAP activation in macrophages. Mol. Cell. Biol., IS: 3579-3586, 16. Phan-Bich, L., Buard, A., Petit, J-F., Zeng, L.. Tenu, J-P., ChrÃ©tien,P., Monnet, I., 1995. Boutin, C., Bignon, J., ternaire. G., and Jaurand, M-C. Differential responsiveness of 35. Wen, Z.. Zhong, Z.. and Darnell, J. E. Maximal activation of transcription by Stall human and rat mesothelioma cell lines to recombinant interferon-y Am. J. Respir. and Stat3 requires both tyrosine and serine phosphorylation. Cell, 82: 241-250, 1995. Cell Mol. Biol., 16: 178-186, 1997. 36. Zhang. X., Blenis, J., Li, H-C, Schindler, C., and Chen-Kiang. S. Requirement of 17. MÃ¼ller,M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A.. Silvennoinen, O.. serine phosphorylation for formation of STAT-promoter complexes. Science (Wash Harpur, A. G., Barbieri, G., Witthuhn, B. A., Schindler, C., Pellegrini, S., Wilks, ington DC), 267: 1990-1994. 1995. A. F., Ihle, J. N., Stark, G. R., and Kerr, I. M. The protein tyrosine kinase JAK1 37. Xu, X., Sun, Y-L., and Hoey, T. Cooperative DNA binding and sequence-selective complements defects in interferon-o//3 and -y signal transduction. Nature (Lond.), 366: 129-135, 1993. recognition conferred by the STAT amino-terminal domain. Science (Washington DC). 273: 794-797. 1996. 18. Watling, D., Guschin, D., MÃ¼ller,M., Silvennoinen, O., Witthuhn, B. A., Quelle, 38. Leaman, D. W.. Leung. S., Li, X., and Stark, G. R. Regulation of STAT-dependent F. W., Rogers, N. C., Schindler, C., Stark, G. R.. Ihle, J. N., and Kerr, I. M. pathways by growth factors and cytokines. FASEB J., 10: 1578-1588, 1996. Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-y signal transduction pathway. Nature (Lond.), 366: 166-170. 1993. 39. Gerwin. B. Mesothelial carcinogenesis. Possible avenues of growth factor promotion. 19. Schindler, C., and Darnell, J. E. Transcriptional responses to polypeptide ligands: the In: M-C. Jaurand and J. Bignon (eds.). The Mesothelial Cell and Mesothelioma, Vol. JAK-STAT pathway. Annu. Rev. Biochem., 64: 621-651, 1995. 78. pp. 223-243. New York: Marcel Dekker, Inc., 1994. 20. Durbin, J. E., Hackenmiller, R., Simon, M. C., and Levy, D. E. Targeted disruption 40. Fitzpatrick. D. R., Peroni, D. J., and Bielefeldt-Ohmann. H. The role of growth factors of the mouse Stall gene results in compromised innate immunity to viral disease. and cytokines in the tumorigenesis and immunobiology of malignant mesothelioma. Cell, 84: 443-450, 1996. Am. J. Respir. Cell Mol. Biol., 12: 455-460, 1995.

847

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. Human Malignant Mesothelioma Cell Growth: Activation of Janus Kinase 2 and Signal Transducer and Activator of Transcription 1α for Inhibition by Interferon-γ

Annie Buard, Claire Vivo, Isabelle Monnet, et al.

Cancer Res 1998;58:840-847.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/4/840

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/58/4/840. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.